The goal of the proposed research is to understand the mechanisms by which NO and TNFalpha regulate CGRP expression in cultured trigeminal neurons and an in vivo model of TMJ inflammation. CGRP is thought to play a key role in underlying pathology of TMJ disorders (TMJDs). High levels of the neuropeptide CGRP present in the synovial fluid during TMJDs are associated with pain. In addition to CGRP, the levels of NO and TNF have been reported to be elevated during TMJ inflammation. The cellular mechanisms by which CGRP expression is controlled during TMJDs are not known. In this proposal, I will test the hypothesis that NO and TNF directly stimulate CGRP gene expression in cultured trigeminal ganglia neurons and in vivo TMJ models. In addition, the role of NO and TNF in the pathogenesis of TMJ inflammation will be investigated. Studies proposed in the first aim will determine the effect of NO and TNF alone or in combination with other inflammatory mediators on CGRP release from trigeminal neurons. The second aim will focus on identifying the basal (unstimulated), and NO- and TNF-responsive regulatory sites in the CGRP promoter. Primary trigeminal cultures will be transiently transfected with CGRP-luciferase reporter constructs and reporter activity measured. The goal of the third aim is to elucidate the pathways involved in NO and TNF signaling in trigeminal neurons. Specific cyclase and kinase inhibitors and activators will be used to identify the major signaling pathway(s) involved in regulating CGRP. Further studies of individual pathways will utilize phosphospecific antibodies and signaling pathway detection kits. In the fourth aim, NO and TNF regulation of CGRP expression will be extended to in vivo models of TMJ inflammation. Also, the role of NO and TNF in the pathogenesis of TMJ inflammation in vivo will be investigated and whether specific inhibitors of signaling pathways can reduce TMJ inflammation. Results from these studies will provide valuable insight into the cellular and molecular mechanisms that control CGRP gene expression in trigeminal neurons that may lead to the development of novel therapeutic strategies for TMJDs and other inflammatory diseases.